This invention relates to a method for the separation of blood plasma proteins from cell culture systems such as, for example, fermentation broths of microbial cell cultures and expended media of mammalian cell cultures.
The fractional separation of the various species of proteins which occur in human plasma or serum has been the concern of scientists in the medical and pharmaceutical fields for many years. A significant part of this interest involves investigations for isolating the plasma components responsible for the clotting of blood, that is the blood coagulation factors. Other blood protein components of great medical interest are the gamma globulins, which are the carriers of antibody activity, and albumin, which is the main regulator of the colloid-osmotic pressure of plasma.
The most important of these plasma protein species are today harvested on an industrial scale with the natural source material being human donor blood. The importance of the isolation of blood plasma protein species is readily illustrated by the need for a commercial supply of antihemophilic factor (AHF; Factor VIII). The criticality of Factor VIII in hemostasis and blood coagulation is well-known. Most patients with a congenital clotting disorder have a Factor VIII deficiency (hemophilia A patients) while a lesser number have Factor IX deficiency (hemophilia B patients) or other minor coagulation factor deficiencies. Patients suffering from such clotting deficiencies have in the past relied upon transfusions of whole blood plasma or, preferably, on administration of plasma concentrates which have been purified to contain higher levels of Factor VIII or Factor IX. Cryoprecipitates such as developed by Judith Pool, New Eng. J. Med. 274, 1443-47 (1965), or still more concentrated fractions such as Hemofil.RTM. AHF, produced by methods described in U.S. Pat. Nos. 3,415,804, 3,631,018, 4,089,944 and Re. 29,698, are typical examples of commercially available fractions having high levels of Factor VIII. These commercially produced fractions depend, however, on the availability of a scarce commodity, namely a limited supply of donor blood.
With recently developed techniques of genetic engineering and molecular biology applied to the traditional processes of industrial microbiology, it is now feasible to produce mammalian plasma proteins in microbial species. Thus, the principal architects of gene splicing have disclosed the applicability of their methodology to the production of various plasma proteins such as antihemophilia protein, gamma globulins, albumin, fibrinogen and prothrombin. See Cohen and Boyer, U.S. Pat. No. 4,237,224, col.9. While the actual early work in genetic engineering was limited to the production of relatively small proteins such as somatostatin and insulin, the basic principles of gene splicing for the production of foreign proteins in bacteria and yeasts have now been shown to be adaptable to the preparation of much larger proteins. Thus, relatively large proteins which have already been produced by recombinant microorganisms are for example, ovalbumin having a molecular weight of about 43,000, as described in Ger. Offen. Nos. 2,923,297 and 2,933,000 and in Fr. Demande 2,458,585 and 2,476,126, and human serum albumin which has a chain of 585 amino acids, as described by Lawn et al., Nucleic Acids Res. 9 (22), 6103-6114 (1981).
The need to develop a useful method for the separation of plasma proteins from cell culture systems has now been recognized by the present inventor. The separation of any specific desired protein from admixture with other proteins and cell constituents, metabolites, cell debris and the like substances which are present in fermentation broths and expended media entails major difficulties. In the past, the recovery and isolation of protein products from fermentation broths and expended media has involved a variety of procedures such as:
Extraction by liberation from cells or cellular constituents by mechanical, physical or chemical disruption of the cell wall or membrane, for example, by use of homogenizers or by use of aqueous and organic solvent extraction;
Precipitation by salting out with salts such as (NH.sub.4).sub.2 SO.sub.4, or by use of organic solvents such as ethanol, methanol or isopropanol, or high molecular weight polymers such as polyethylene glycol and dextran, or with metal ions and complexes, or by use of differential temperature and pH conditions;
Adsorption with colloidal materials such as bentonite, calcium phosphate, barium sulfate, hydroxyapetite, activated carbon, silica or Al(OH).sub.3 gels;
Centrifugation, filtration with or without filteraids such as kieselguhr and other such diatomaceous earths, or ultrafiltration;
Chromatography with gel filtration and ion-exchange resins such as with Sephadex.RTM. (cross-linked dextran) gels, Sepharose.RTM. (agarose) gels, and DEAE-Sephadex or DEAE-cellulose ion exchange resins;
Electrophoresis;
Ultracentrifugation; and
Finishing operations such as desalting, concentration and drying.
A more recently developed method for recovery and isolation of proteins involves immunoaffinity chromatography. Monoclonal antibodies having an affinity for a particular protein can be attached to polysaccharide beads which are placed in a chromatographic column. When the crude protein solution is passed through the column, the desired protein molecules are adsorbed on the beads while the impurities or undesired materials pass through the column. The desired protein is then eluted from the beads by adjustment in pH with a suitable washing solution.